Prevalence of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency among Malaria Patients in Southern Thailand: 8 Years Retrospective Study.

Erythrocytes deficient in glucose-6-phosphate dehydrogenase (G6PD) is more susceptible to oxidative damage from free radical derived compounds. The hemolysis triggered by oxidative agents such as primaquine (PQ) is used for the radical treatment of hypnozoites of P. vivax. Testing of G6PD screening before malaria treatment is not a common practice in Thailand, which poses patients at risk of hemolysis. This retrospective study aimed to investigate the prevalence of G6PD in malaria patients who live in Southern Thailand. Eight hundred eighty-one malaria patients were collected for 8-year from 2012 to 2019, including 785 (89.1%) of P. vivax, 61 (6.9%) of P. falciparum, 27 (3.1%) of P. knowlesi, and 8 (0.9%) of mixed infections. The DiaPlexC genotyping kit (Asian type) and PCR-RFLP were employed to determine the G6PD variants. The result showed that 5 different types of G6PD variants were identified in 26 cases (2.9%); 12/26 (46.2%) had Mahidol (487G>A) and 11/26 (42.3%) had Viangchan (871G>A) variants, while the rest had Kaiping (1388G>A), Union (1360C>T), and Mediterranean (563C>T) variants. G6PD Songklanagarind (196T>A) variant was not found in the study. Our result did not show a significant difference in the malaria parasite densities in patients between G6PD-deficient and G6PD-normal groups. According to our findings, testing G6PD deficiency and monitoring the potential PQ toxicity in patients who receive PQ are highly recommended.

A LAMP-SNP Assay Detecting C580Y Mutation in Pfkelch13 Gene from Clinically Dried Blood Spot Samples.

Artemisinin resistance (ART) has been confirmed in Greater Mekong Sub-region countries. Currently, C580Y mutation on Pfkelch13 gene is known as the molecular marker for the detection of ART. Rapid and accurate detection of ART in field study is essential to guide malaria containment and elimination interventions. A simple method for collection of malaria-infected blood is to spot the blood on filter paper and is fast and easy for transportation and storage in the field study. This study aims to evaluate LAMP-SNP assay for C580Y mutation detection by introducing an extra mismatched nucleotide at the 3' end of the FIP primer. The LAMP-SNP assay was performed in a water bath held at a temperature of 56°C for 45 min. LAMP-SNP products were interpreted by both gel-electrophoresis and HNB-visualized changes in color. The method was then tested with 120 P. falciparum DNA from dried blood spot samples. In comparing the LAMP-SNP assay results with those from DNA sequencing of the clinical samples, the 2 results fully agreed to detect C580Y. The sensitivity and specificity of the LAMP-SNP assay showed 100%. There were no cross-reactions with other Plasmodium species and other Pfkelch13 mutations. The LAMP-SNP assay performed in this study was rapid, reliable, and useful in detecting artemisinin resistance in the field study.

Outer membrane protein A (OmpA) is a potential virulence factor of Vibrio alginolyticus strains isolated from diseased fish.

Vibrio alginolyticus is one of the most serious causative agents of diseases in cultured marine fish and shellfish. However, the characteristics of virulence factors in pathogenic V. alginolyticus are poorly known. To gain insight into fish diseases caused by V. alginolyticus, we carried out two-dimensional gel electrophoresis (2-DE) combined with MALDI-TOF mass spectrometry to identify uniquely expressed proteins in the disease-causing V. alginolyticus. V. alginolyticus strains were isolated from marine environments and diseased fish obtained from southern Thailand. We identified seven unique proteins in the disease-causing V. alginolyticus strain. Among those, the outer membrane protein A (OmpA) had the strongest expression. Therefore, the function of this protein was further analysed. To investigate the role of OmpA protein, an in-frame deletion mutant of ompA was constructed using the homologous recombination method. Although the ompA mutant V. alginolyticus strain (ΔompA) grew normally, the mutant exhibited a significant defect in the swarming ability and the biofilm formation. Furthermore, Galleria mellonella larvae injected with the mutant bacteria had a significantly greater survival percentage than those injected with the wild-type strain, demonstrating that OmpA protein is required for the pathogenicity of V. alginolyticus. Together, this study suggests a potential target for vaccine development against pathogenic V. alginolyticus strain.

Molecular detection of drug resistant malaria in Southern Thailand.

BACKGROUND: Drug resistance within the major malaria parasites Plasmodium vivax and Plasmodium falciparum threatens malaria control and elimination in Southeast Asia. Plasmodium vivax first-line treatment drug is chloroquine together with primaquine, and the first-line treatment for P. falciparum malaria is artemisinin in combination with a partner drug. Plasmodium vivax and P. falciparum parasites resistant to their respective first-line therapies are now found within Southeast Asia. The resistance perimeters may include high transmission regions of Southern Thailand which are underrepresented in surveillance efforts. METHODS: This study investigated blood samples from malaria centres in Southern Thailand. Genetic loci associated with drug resistance were amplified and sequenced. Drug resistance associated genes Pvmdr1, Pvcrt-o, Pvdhfr, and Pvdhps were characterized for 145 cases of P. vivax malaria, as well as the artemisinin resistance-associated Pfkelch13 gene from 91 cases of P. falciparum malaria. RESULTS: Plasmodium vivax samples from Southern Thai provinces showed numerous chloroquine and antifolate resistance-associated mutations, including SNP and Pvcrt-o K10-insertion combinations suggestive of chloroquine resistant P. vivax phenotypes. A high proportion of the C580Y coding mutation (conferring artemisinin resistance) was detected in P. falciparum samples originating from Ranong and Yala (where the mutation was previously unreported). CONCLUSIONS: The results demonstrate a risk of chloroquine and antifolate resistant P. vivax phenotypes in Southern Thailand, and artemisinin resistant P. falciparum observed as far south as the Thai-Malaysian border region. Ongoing surveillance of antimalarial drug resistance markers is called for in Southern Thailand to inform case management.

Genetic Diversity of Plasmodium vivax in Clinical Isolates from Southern Thailand using PvMSP1, PvMSP3 (PvMSP3α, PvMSP3ß) Genes and Eight Microsatellite Markers.

Plasmodium vivax is usually considered morbidity in endemic areas of Asia, Central and South America, and some part of Africa. In Thailand, previous studies indicated the genetic diversity of P. vivax in malaria-endemic regions such as the western part of Thailand bordering with Myanmar. The objective of the study is to investigate the genetic diversity of P. vivax circulating in Southern Thailand by using 3 antigenic markers and 8 microsatellite markers. Dried blood spots were collected from Chumphon, Phang Nga, Ranong and, Surat Thani provinces of Thailand. By PCR, 3 distinct sizes of PvMSP3α, 2 sizes of PvMSP3ß and 2 sizes of PvMSP1 F2 were detected based on the length of PCR products, respectively. PCR/RFLP analyses of these antigen genes revealed high levels of genetic diversity. The genotyping of 8 microsatellite loci showed high genetic diversity as indicated by high alleles per locus and high expected heterozygosity (HE). The genotyping markers also showed multiple-clones of infection. Mixed genotypes were detected in 4.8% of PvMSP3α, 29.1% in PvMSP3ß and 55.3% of microsatellite markers. These results showed that there was high genetic diversity of P. vivax isolated from Southern Thailand, indicating that the genetic diversity of P. vivax in this region was comparable to those observed other areas of Thailand.

Molecular Surveillance of Pfkelch13 and Pfmdr1 Mutations in Plasmodium falciparum Isolates from Southern Thailand.

Artemisinin-based combination therapy (ACT) resistance is widespread throughout the Greater Mekong Subregion. This raises concern over the antimalarial treatment in Thailand since it shares borders with Cambodia, Laos, and Myanmar where high ACT failure rates were reported. It is crucial to have information about the spread of ACT resistance for efficient planning and treatment. This study was to identify the molecular markers for antimalarial drug resistance: Pfkelch13 and Pfmdr1 mutations from 5 provinces of southern Thailand, from 2012 to 2017, of which 2 provinces on the Thai- Myanmar border (Chumphon and Ranong), one on Thai-Malaysia border (Yala) and 2 from non-border provinces (Phang Nga and Surat Thani). The results showed that C580Y mutation of Pfkelch13 was found mainly in the province on the Thai-Myanmar border. No mutations in the PfKelch13 gene were found in Surat Thani and Yala. The Pfmdr1 gene isolated from the Thai-Malaysia border was a different pattern from those found in other areas (100% N86Y) whereas wild type strain was present in Phang Nga. Our study indicated that the molecular markers of artemisinin resistance were spread in the provinces bordering along the Thai-Myanmar, and the pattern of Pfmdr1 mutations from the areas along the international border of Thailand differed from those of the non-border provinces. The information of the molecular markers from this study highlighted the recent spread of artemisinin resistant parasites from the endemic area, and the data will be useful for optimizing antimalarial treatment based on regional differences.

Acanthamoeba in Southeast Asia - Overview and Challenges.

Acanthamoeba, one of free-living amoebae (FLA), remains a high risk of direct contact with this protozoan parasite which is ubiquitous in nature and man-made environment. This pathogenic FLA can cause sight-threatening amoebic keratitis (AK) and fatal granulomatous amoebic encephalitis (GAE) though these cases may not commonly be reported in our clinical settings. Acanthamoeba has been detected from different environmental sources namely; soil, water, hot-spring, swimming pool, air-conditioner, or contact lens storage cases. The identification of Acanthamoeba is based on morphological appearance and molecular techniques using PCR and DNA sequencing for clinico-epidemiological purposes. Recent treatments have long been ineffective against Acanthamoeba cyst, novel anti-Acanthamoeba agents have therefore been extensively investigated. There are efforts to utilize synthetic chemicals, lead compounds from medicinal plant extracts, and animal products to combat Acanthamoeba infection. Applied nanotechnology, an advanced technology, has shown to enhance the anti-Acanthamoeba activity in the encapsulated nanoparticles leading to new therapeutic options. This review attempts to provide an overview of the available data and studies on the occurrence of pathogenic Acanthamoeba among the Association of Southeast Asian Nations (ASEAN) members with the aim of identifying some potential contributing factors such as distribution, demographic profile of the patients, possible source of the parasite, mode of transmission and treatment. Further, this review attempts to provide future direction for prevention and control of the Acanthamoeba infection.

CHARACTERIZATION OF MALARIA INFECTION AT TWO BORDER AREAS OF THAILAND ADJOINING WITH MYANMAR AND MALAYSIA.

During 2009 to 2010, a total of 408 blood samples collected from malaria patients in Ranong (149) and Yala (259) Provinces, Thailand were investigated for Plasmodium spp using microscopic examination. There are no statistical differences in the prevalence of P. falciparum and P. vivax in samples collected from Ranong and Yala (46% vs 52%, and 54% vs 45%, respectively). Single nucleotide polymorphism of codon 86 in pfmdr1 (encoding P. falciparum multidrug resistance protein 1) was investigated among 75 samples of P. falciparum and 2 samples of P. knowlesi. A pfmdr1 N86Y mutation was detected in 1 out of 29 samples and 45 out of 46 samples obtained from Ranong and Yala Provinces, respectively. It is interesting that pfmdr1 was detected in two P. knowlesi DNA samples obtained previously from Ranong Province which was 99% homologous to pfmdr1 obtained from falciparum parasites in the same area but the mutation was not observed. The difference in multidrug resistance protein in Plasmodium obtained from those two border areas of Thailand will be of use in monitoring drug resistance in these border regions of the country.

Toxoplasma gondii infection: What is the real situation?

The prevalence of chronic Toxoplasma infections reported in the literature varies enormously. We hypothesize that one factor could be due to the different methods used in the evaluation of infections. Serological evidence of Toxoplasma infections in 450 pregnant women (PW) and 300 HIV-infected patients (HIV) were investigated by the Sabin-Feldman dye test and two other commercial ELISA kits (kit1 and kit2). Anti-Toxoplasma IgG antibodies obtained from the Sabin-Feldman dye test, ELISA kit1 and ELISA kit2 in the PW subjects were 14.7%, 29.6% and 38.7%, and in the HIV subjects were 13%, 34.7% and 36.3%, respectively. So there were significant differences in the seroprevalences when different diagnostic tests were used (P<0.05). Regarding Sabin-Feldman dye test as the gold standard for anti-Toxoplasma antibodies detection, we found that the sensitivity and specificity of the ELISA kit1 and kit2 was in the range of their specification. However as the two ELISA kits used in our study identified a much higher prevalence of Toxoplasma infections which indicated that false positive cases were being reported. Based on results obtained, it is therefore highly recommended that research workers should be aware that the reports of serological studies in terms of high positive results should be treated with some skepticism until additional precise diagnostic tools are developed.

Waterborne parasites and physico-chemical assessment of selected lakes in Malaysia.

The objective of this study was to assess the physico-chemical parameters and waterborne parasites in selected recreational lakes from Malaysia. Samples were collected from seven stations of Recreational Lake A (RL-A) and six stations of Recreational Lake B (RL-B). The samples were processed to detect the presence of Giardia spp. and Cryptosporidium spp. using immunomagnetic separation kit, helminth eggs or ova by bright field microscopy and Acanthamoeba spp. by cultivation in non-nutrient agar. Chemical parameters such as ammonia, chlorine, fluoride, nitrate and nitrite and physical parameters such as dissolved oxygen, electrical conductivity, pH, salinity, temperature and total dissolved solid were also measured. Both lakes were freshwater with salinity ranging from 0.05 to 0.09 ppt. Most stations of these lakes were contaminated with Cryptosporidium spp., Giardia spp., Ascaris spp. and hookworm. Schistosoma spp. was found in RL-B only, while Acanthamoeba spp. was found in all stations. Of all sampling sites, station 5 of RL-B is the most contaminated. Linear regression and correlation analysis revealed that Giardia spp. and Schistosoma spp. showed a significant negative correlation with turbidity (p < 0.01). Based on the preliminary data obtained, it is clearly shown that there is a necessity to implement the detection of waterborne parasites and physico-chemical analysis in Malaysia. Future work on heavy metals (chromium, copper, mercury and zinc) is recommended to enhance the overall water quality monitoring and to take appropriate safety measures to ensure maintenance of good water standards.

Application of loop-mediated isothermal amplification combined with lateral flow assay visualization of Plasmodium falciparum kelch 13 C580Y mutation for artemisinin resistance detection in clinical samples.

Resistance to the antimalarial drug artemisinin (ART) has emerged in Greater Mekong Subregion. The molecular marker predominantly used to identify ART resistance is the C580Y mutation in Pfkelch13 of Plasmodium falciparum. Rapid and accurate detection of ART resistance in the field is necessary to guide malaria containment and elimination interventions. Our study evaluates the PfC580Y by using the loop-mediated isothermal amplification and single nucleotide polymorphism analysis visualization using a lateral flow assay (LAMP-SNP-LFA) method for detecting ART resistance in clinical samples collected from Thailand between 2014 and 2019. The optimized incubation condition for the reaction was determined as 45 min at 56 °C, followed by visual detection of positive amplicons using LFA. The assay demonstrated high analytical sensitivity and specificity, with a limit of detection of 16.8 copies of C580Y plasmid/µL of and 100% accuracy for C580Y mutation detection. The PfC580Y LAMP-SNP-LFA method is faster and simpler than conventional polymerase chain reaction/DNA sequencing and has the potential to support antimalarial management policies, malaria control, and global elimination efforts.

Human Plasmodium knowlesi infection in Ranong province, southwestern border of Thailand.

BACKGROUND: Plasmodium knowlesi, a simian malaria parasite, has been reported in humans in many Southeast Asian countries. In Thailand, most of the limited numbers of cases reported so far were from areas near neighbouring countries, including Myanmar. METHODS: Blood samples collected from 171 Thai and 248 Myanmese patients attending a malaria clinic in Ranong province, Thailand, located near the Myanmar border were investigated for P. knowlesi using nested PCR assays. Positive samples were also investigated by PCR for Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale, and were confirmed by sequencing the gene encoding the circumsporozoite protein (csp). RESULTS: Two samples, one obtained from a Thai and the other a Myanmese, were positive for P. knowlesi only. Nucleotide sequences of the csp gene derived from these two patients were identical and phylogenetically indistinguishable from other P. knowlesi sequences derived from monkeys and humans. Both patients worked in Koh Song, located in the Kawthoung district of Myanmar, which borders Thailand. CONCLUSION: This study indicates that transmission of P. knowlesi is occurring in the Ranong province of Thailand or the Kawthoung district of Myanmar. Further studies are required to assess the incidence of knowlesi malaria and whether macaques in these areas are the source of the infections.

Development of a highly sensitive nucleic acid amplification-based detection for human leptospirosis infection.

Highly sensitive diagnostic tools are crucial for individual screening during an epidemic of leptospirosis. To aid in developing a diagnostic tool for the sensitive detection of pathogenic strains, a new approach targeting nucleic acid amplification that combines quantitative PCR (qPCR) and strand displacement isothermal amplification was evaluated. The effectiveness of the combined approach, a quantitative polymerase chain displacement reaction (qPCDR), was compared with a qPCR technique. The results showed that qPCDR presented higher sensitivity (at least tenfold) and shorter reaction time than the qPCR approach for pathogenic Leptospira spp. detection. Thus, the qPCDR-based technique developed in this study is a promising approach for pathogenic Leptospira spp. detection and the further development of a diagnostic kit.

Parasitic infections in HIV infected individuals: diagnostic & therapeutic challenges.

After 30 years of the human immunodeficiency virus (HIV) epidemic, parasites have been one of the most common opportunistic infections (OIs) and one of the most frequent causes of morbidity and mortality associated with HIV-infected patients. Due to severe immunosuppression, enteric parasitic pathogens in general are emerging and are OIs capable of causing diarrhoeal disease associated with HIV. Of these, Cryptosporidium parvum and Isospora belli are the two most common intestinal protozoan parasites and pose a public health problem in acquired immunodeficiency syndrome (AIDS) patients. These are the only two enteric protozoan parasites that remain in the case definition of AIDS till today. Leishmaniasis, strongyloidiasis and toxoplasmosis are the three main opportunistic causes of systemic involvements reported in HIV-infected patients. Of these, toxoplasmosis is the most important parasitic infection associated with the central nervous system. Due to its complexity in nature, toxoplasmosis is the only parasitic disease capable of not only causing focal but also disseminated forms and it has been included in AIDS-defining illnesses (ADI) ever since. With the introduction of highly active anti-retroviral therapy (HAART), cryptosporidiosis, leishmaniasis, schistosomiasis, strongyloidiasis, and toxoplasmosis are among parasitic diseases reported in association with immune reconstitution inflammatory syndrome (IRIS). This review addresses various aspects of parasitic infections in term of clinical, diagnostic and therapeutic challenges associated with HIV-infection.

A colorimetric method for the evaluation of anti-giardial drugs in vitro.

The aim of this study was to develop a simple and reliable method to determine the viability of Giardia intestinalis after incubation with an anti-giardial agent by using a colorimetric method. Factors that may affect the optical density value were systematically evaluated. The most suitable conditions were obtained when G. intestinalis trophozoites, 5 × 10(5)cells/ml were incubated with the anti-giardial agent for 48 h. The culture medium was removed and trophozoites were immediately fixed by immersing the whole plate in absolute methanol for 2 min. The fixed trophozoites were then stained with 0.1% w/v methylene blue for 10 min, washed once by immersing the whole plate into distilled water. The dye was released by adding 0.1M hydrochloric acid solution (300 µl) and the optical density was read at 655 nm. The 50% inhibitory concentration values (IC(50)) of metronidazole, ornidazole and furazolidone obtained from our proposed method (0.41 ± 0.06, 0.18 ± 0.01, 0.26 ± 0.13 µg/ml, respectively) were comparable to the IC(50) values obtained by the current conventional method (0.14 ± 0.05, 0.15 ± 0.04, 0.14 ± 0.02 µg/ml, respectively). This new method did provide a convenient and reliable way to screen for potential anti-giardial agents.

Molecular detection of antimalarial drug resistance in Plasmodium vivax from returned travellers to NSW, Australia during 2008-2018.

To monitor drug resistance in Plasmodium vivax, a multidrug resistance 1 (Pvmdr1) gene and a putative transporter protein (Pvcrt-o) gene were used as molecular markers for chloroquine resistance. The biomarkers, the dihydrofolate reductase (Pvdhfr) gene and the dihydropteroate synthetase (Pvdhps) gene, were also used for the detection of resistance to sulphadoxine-pyrimethamine (SP); this drug is often accidentally used to treat P. vivax infections. Clinical blood samples (n = 120) were collected from patients who had been to one of eight malaria-endemic countries and diagnosed with P. vivax infection. The chloroquine resistance marker, the Pvmdr1 gene, showed F976:L1076 mutations and L1076 mutation. A K10 insertion in the Pvcrt-o gene was also found among the samples successfully sequenced. A combination of L/I57:R58:M61:T117 mutations in the Pvdhfr gene and G383:G553 mutations in the Pvdhps gene were also observed. Mutations found in these genes indicate that drug resistance is present in these eight countries. Whether or not countries are using chloroquine to treat P. vivax, there appears to be an increase in mutation numbers in resistance gene markers. The detected changes in mutation rates of these genes do suggest that there is still a trend towards increasing P. vivax resistance to chloroquine. The presence of the mutations associated with SP resistance indicates that P. vivax has had exposure to SP and this may be a consequence of either misdiagnosis or coinfections with P. falciparum in the past.

Molecular epidemiology of resistance to antimalarial drugs in the Greater Mekong subregion: an observational study.

BACKGROUND: The Greater Mekong subregion is a recurrent source of antimalarial drug resistance in Plasmodium falciparum malaria. This study aimed to characterise the extent and spread of resistance across this entire region between 2007 and 2018. METHODS: P falciparum isolates from Myanmar, Thailand, Laos, and Cambodia were obtained from clinical trials and epidemiological studies done between Jan 1, 2007, and Dec 31, 2018, and were genotyped for molecular markers (pfkelch, pfcrt, pfplasmepsin2, and pfmdr1) of antimalarial drug resistance. Genetic relatedness was assessed using microsatellite and single nucleotide polymorphism typing of flanking sequences around target genes. FINDINGS: 10 632 isolates were genotyped. A single long pfkelch Cys580Tyr haplotype (from -50 kb to +31·5 kb) conferring artemisinin resistance (PfPailin) now dominates across the eastern Greater Mekong subregion. Piperaquine resistance associated with pfplasmepsin2 gene amplification and mutations in pfcrt downstream of the Lys76Thr chloroquine resistance locus has also developed. On the Thailand-Myanmar border a different pfkelch Cys580Tyr lineage rose to high frequencies before it was eliminated. Elsewhere in Myanmar the Cys580Tyr allele remains widespread at low allele frequencies. Meanwhile a single artemisinin-resistant pfkelch Phe446Ile haplotype has spread across Myanmar. Despite intense use of dihydroartemisinin-piperaquine in Kayin state, eastern Myanmar, both in treatment and mass drug administrations, no selection of piperaquine resistance markers was observed. pfmdr1 amplification, a marker of resistance to mefloquine, remains at low prevalence across the entire region. INTERPRETATION: Artemisinin resistance in P falciparum is now prevalent across the Greater Mekong subregion. In the eastern Greater Mekong subregion a multidrug resistant P falciparum lineage (PfPailin) dominates. In Myanmar a long pfkelch Phe446Ile haplotype has spread widely but, by contrast with the eastern Greater Mekong subregion, there is no indication of artemisinin combination therapy (ACT) partner drug resistance from genotyping known markers, and no evidence of spread of ACT resistant P falciparum from the east to the west. There is still a window of opportunity to prevent global spread of ACT resistance. FUNDING: Thailand Science Research and Innovation, Initiative 5%, Expertise France, Wellcome Trust.

Genotyping of Toxoplasma gondii Isolated from cat Feces in Songkhla, Southern Thailand.

The genotype of Toxoplasma gondii has been extensively studied to determine whether its characteristics may influence the course of toxoplasmosis. Recent genotyping studies in South America have found Toxoplasma hybrid isolates or atypical strains which were more virulent and highly diverse than North American and European species. Although, the high seroprevalence of Toxoplasma infection among pregnant women and HIV-infected patients from Songklanagarind Hospital, Songkhla, Southern Thailand has been previously reported, there is no reported study of human genotyping data on T. gondii strains. This is the first genetic typing of T. gondii isolates obtained from naturally infected cats in Thailand. Five chromosomal loci were analyzed by a nested PCR-RFLP technique to determine the genotype of 13 T. gondii isolates from cat feces gathered in the South of Thailand. The PCR-RFLP patterns of SAG1, SAG2-new, SAG3, BTUB and GRA6 markers resulted in five diverse genotypes: the type I (one isolate), type III (two isolates), type II or type III (one isolate), recombinant genotypes (two isolates) and atypical genotypes (two isolates). The presence of unusual genotypes may lead to new virulent traits associated with more severe forms of human Toxoplasma infections. More evaluations are needed before conclusions could be made as to whether the oocysts from cat feces play an important role in the severity of Toxoplasma infections.

Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries.

Data on the distribution of free-living amoebae is still lacking especially in Southeast Asian region. The aquatic environment revealed a high occurrence of free-living amoebae (FLA) due to its suitable condition and availability of food source, which subsequently causes infection to humans. A total of 94 water samples consisted of both treated and untreated from Laos (31), Myanmar (42), and Singapore (21) were investigated for the presence of pathogenic FLA. Each water sample was filtered and cultured onto non-nutrient agar seeded with live suspension of Escherichia coli and incubated at room temperature. Morphological identification was conducted for both trophozoites and cysts via microscopic stains (Giemsa and immunofluorescence). The presence of Naegleria-like structures was the most frequently encountered in both treated and untreated water samples, followed by Acanthamoeba-like and Vermamoeba-like features. To identify the pathogenic isolates, species-specific primer sets were applied for molecular identification of Acanthamoeba, Naegleria, and Vermamoeba. The pathogenic species of Acanthamoeba lenticulata and A. triangularis were detected from untreated water samples, while Vermamoeba vermiformis was found in both treated and untreated water samples. Our results suggested that poor water quality as well as inadequate maintenance and treatment might be the cause of this alarming problem since chlorine disinfection is ineffective in eradicating these amoebas in treated water samples. Regular monitoring and examination of water qualities are necessary in order to control the growth, hence, further preventing the widespread of FLA infections among the public.

Correction: Pathogenic waterborne free-living amoebae: An update from selected Southeast Asian countries.

[This corrects the article DOI: 10.1371/journal.pone.0169448.].